MECHANISMS OF OCULAR PENETRATION ENHANCERS

IV. MECHANISMS OF OCULAR PENETRATION ENHANCERS

A detailed mechanistic description of penetration enhancement is beyond the scope of this chapter and can be found elsewhere (13). Our main focus is on ocular penetration enhancement (14). Ideally, penetration enhancers should have the following characteristics (15):

1. The absorbing-enhancing action should be immediate and uni- directional, and the duration should be specific and predictable.

2. There is immediate recovery of the tissue after removing the absorption enhancers.

3. There is no systemic and local effect associated with the enhancers.

288 Lee and Robinson

4. The enhancers should be physically and chemically compatible with a wide range of drugs and excipients.

However, currently available penetration enhancers are far from satisfying the above requirements. None have yet been approved by the FDA presum- ably because of safety concerns. In order to design an efficient and safe penetration enhancer, it is necessary to have a thorough understanding of the mechanisms of penetration enhancement. Basically, penetration enhan- cers work by one or more of the following mechanisms (13):

1. Altering membrane structure and enhancing transcellular trans- port by extracting membrane components and/or increasing fluidity.

2. Enhancing paracellular transport: Chelating calcium ions leads to opening of tight junctions; Inducing high osmotic pressure that transiently opens tight junctions; Introducing agents to disrupt the structure of tight junctions.

3. Altering mucus structure and rheology so that this diffusion bar- rier is weakened

4. Modifying the physical properties of the drug-enhancer entity

5. Inhibiting enzyme activity

A summary of ocular penetration enhancers is shown in Table 4 (14). Typically, ocular penetration enhancement falls into two categories: para- cellular and transcellular.

A. Enhanced Paracellular Transport As mentioned earlier, tight junctions are the major determinant of paracel-

lular transport. In other words, tight junctions are the primary targets for a penetration enhancer to act on in order to improve paracellular transport. The most well-known penetration enhancer to improve paracellular trans- port is EDTA, which is a calcium chelator commonly used as a preservative. It is well known that proper functioning of tight junctions depends on calcium ions. In the absence of calcium ions, there is a widening of tight junctions, resulting in an increase in paracellular permeability (8). EDTA can remove divalent ions by its chelating action. Therefore, there is no surprise that it has a permeabilizing effect on biological membranes (16). However, its action on the cornea is believed to be much more complicated. Rojanasakul et al. (17) showed that severe membrane damage is evident in corneas treated with EDTA, bile salts, and surfactants. This disruption of plasma membrane structures by EDTA is somewhat unexpected since it is believed that EDTA only interferes with the ability of calcium to maintain

Table 4 A Summary of Ophthalmic Penetration Enhancers Ocular Enhancers

Effect Surfactants

Penetration Spans 20, 40 and 85,

Enhanced aqueous humor concentration; Tweens 20, 40 and

Tween 20 and Brij 35 at HLB 16–17 are 81, Aptet 100, G

most effective and dose dependent 1045, Brji 35 and 58,

Enhancers Myrj 52 and 53

BL-9

Atenolol, Timolol,

Rabbit

Enhanced Papp 3.4 times for atenolol and

Levobunolol,

7.3 times for timolol

Atenolol, Timolol,

Rabbit

Enhanced Papp 3.9–10.5 times for atenolol

Levobunolol,

and 1.5–3.9 times for timolol

Atenolol, Timolol,

Rabbit

Enhanced Papp 2 times for betaxolol

Levobunlool, Betaxolol

Bile Acids Deoxycholic acid

Atenolol, Timolol,

Rabbit

Enhanced Papp, 1.9 times for atenolol, 5.3

Levobunolol,

times for timolol, 1.4 times for

Betaxolol

levobunlool, and 2.2 times for betaxolol

Enhanced Papp 2.5–8.3 times Taurocholic acid

Atenol, Carteolol,

Rabbit

Enhanced 2.4 times for atenolol, 1.5 times

Tilisolol, Timolol,

for carteolol, 1.4 times for tilisolol, and

Befunolol

2.1 times for timolol

FD-4, FD-10

Rabbit

Enhanced Papp 4.5 times for FD-4 and 289 7.1 times for FD-10

290 Table 4 Continued

Effect

10 mM

6-Carboxyfluorescein

Rabbit

Enhanced penetrated amount 7.2 times

Enhanced penetrated amount slightly Taurodeoxycholic acid

Atenolol, Timolol,

Rabbit

Enhanced Papp 5.8 times for atenolol and

Levobunolol,

1.6 times for timolol

Enhanced Papp 5.2–5.5 times

10 mM

6-Carboxyfluorescein

Rabbit

Enhanced penetrated amount 593 times

Enhanced penetrated amount 30.9–61.5

times

Urodeoxycholic acid

Atenolol, Timolol,

Rabbit

Enhanced Papp 2.1 times for timolol and

Levobunolol,

1.6 times for betaxolol

Betaxolol

Enhanced Papp 8.3–11.0 times Tauroursodeoxycholic

Enhanced Papp 3.0 times for atenolol and acid

Atenolol, Timolol,

Rabbit

Levobunol,

1.5 times for betaxolol

Betaxolol

Lee

Enhanced Papp 3.3 times at 0.1% and Fatty acids

Capric acid

Atenolol, Carteolol,

Rabbit

Enhanced Papp 20.3 times for atenolol, Robinson

Tilisolol, Timolol,

8.9 times for carteolol, 5.1 times for

Befunolol

tilisolol, and 3.0 times for timolol

Effect Ocular

Preservatives Benzalkonium

Enhanced Papp 7.2 times for chloride

Prostaglandin F 2a ,

Pig

Pilocarpine,

prostaglandin F

Penetration 2a , 1.7 times for

Dexamethasone

pilocarpine, and 3.3 times for dexamethasone

Tilisolol, FD-4, FD-

Rabbit

Enhanced Papp 3.5 times for tilisolol, 28.8 10 times for FD-4, and 37.1 times for FD-

Atenolol, Timolol,

Rabbit

Enhanced Papp 5.2 times for atenolol, 2.7

Levobunolol,

times for timolol, and 1.3 times for

FD-4, FD-10

Rabbit

Enhanced Papp 43.6 times for FDA and 60.6 times for FD-10

Increased permeability 4–2.5 times 0.02%

Enhanced miotic response about 20 times

Enhanced the ocular absorption about 80% and the systemic absorption about 40%

Chlorhexidine

Enhanced Papp 1.5 times for digluconate

Enhanced permeability significantly over

Human

at 0.005%

Benzyl alcohol

Tilisolol, FD-4, FD-

Rabbit

Enhanced Papp 2.6 times for FDA and 10 8.1 times for FD-10

Enhanced Papp 1.8 times for pilocarpine

Dexamethasone

and 4.7 times for dexamethasone 291

Table 4 Continued Enhancers

Effect 2-Phenylethanol

Tilisolol, FD-4, FD-

Rabbit

Enhanced Papp 2.7 times for tilisolol, 5.6 10 times for FD-4, and 4.8 times for FD-10

Paraben

Tilisolol, FD-4, FD-

Rabbit

Enhanced Papp 1.9 times for FD-10

Propyl paraben

Enhanced Papp 1.5 times Chelating Agents

Atenolol, Timolol,

Rabbit

Enhanced Papp 1.4 times for atenolol

Levobunolol, Betaxolol

Atenolol, Carteolol,

Rabbit

Enhanced Papp 1.7 times for atenolol, 2.9

Tilisolol, Timolol,

times for carteolol, 2.3 times for timolol,

Befunolol

and 1.6 times for befunolol

FD-4, FD-10

Rabbit

Enhanced Papp 15.5 times for FDA and 39.0 times for FD-10

Atenlool, Timolol,

Rabbit

Enhanced Papp 31 times for atenolol and Lee

Levobunolol,

1.9 times for timolol at 0.5%

Betaxolol

and

Robinson Others

Enhanced ocular and systemic absorption significantly

Enhanced Papp 14.1–87.0 times

Effect Ocular

Enhanced Papp 29.1 times for

Sulfacetamide,

acetazolamide, 16.3 times for

Guanethidine,

guanethidine, >87.3 times for Penetration

Cimetidine,

guanethidine, 31.3 times for cimetidine,

Bunolol,

2.2 times for bunolol, and 2.2. times for

Predisolone,

prednisolone

Flurbiprofen Amide

Enhanced ocular bioavailability 3.9–22.0 times after instillation in rabbits

Enhanced penetration into the cornea and rapidly achieved state-steady state drug level

Hexamethylene

Enhanced Papp 17.4-64.3 times Lauramide Hexamethylene

Enhanced Papp 5.7–100.3 times Octanamide Decylmethylsulfoxide

Enhanced Papp 25–77 times Saponin

Atenolol, Timolol,

Rabbit

Enhanced Papp 16.5 times for atenolol,

Levobunolol,

11.0 times for timolol, 1.3 times for

Betaxolol

levobunolol, 2.0 times for betaxolol

Enhanced Papp 2.1 times at 0.01%, 3.3 times at 0.015%, and 8.3 times at 0.025%

294 Table 4 Continued

Effect

Atenolol, Carteolol,

Rabbit

Enhanced Papp 31.9 times for atenolol,

Tilisolol, Befunolol

13.2 times for carteolol, 7.6 times for tilisolol, 3.3 times for timolol, 2.7 times for befunolol

FD-4, FD-10

Rabbit

Enhanced Papp 100 times for FD-4 and 114 times for FD-10.

FD-4: FITC-dextran (average molecular weight 4400); FD-10: FITC-dextran (average molecular weight 9400); Papp: apparent permeability coefficient. Source : Modified from Ref. 14.

Lee and Robinson

Ocular Penetration Enhancers 295 intercellular integrity, but the effect may be due to concentration and con-

tact time. Nishihata et al. (18) showed that EDTA caused leakage of cell proteins from rectal epithelia. Therefore, there is a possibility that EDTA can exert multiple effects on biological membranes. However, it is believed that its primary action is still on the integrity of tight junctions since it fails to improve delivery of progesterone, which appears to penetrate the cornea primarily by the transcellular route (16).

Cytochalasins are a group of small molecules that bind specifically to actin microfilaments, the major component of the cytoskeleton. It has been shown that the cytoskeleton participates in regulation of epithelial perme- ability in a variety of conditions (19). Therefore, it is a reasonable strategy to design a penetration enhancer to act specifically on the cytoskeleton in order to improve paracellular transport. Rojanaskul et al. (17) showed that cyto- chalasin B decreases TEER of the cornea in a dose-dependent manner. They also studied the safety profile of cytochalasin B in vitro. Confocal micro- scopy showed that cytochalasin B produced negligible damage effect on the cell membrane. Moreover, replacement of cytochalasin B after 30-minute treatment with drug-free GBR results in a complete restoration of TEER, a process that is completed within 30 minutes after solution replacement. However, prolonged exposure time (e.g., > 1 hour) results in permanent damage with incomplete recovery within the time frame of the experiment. It is obvious that cytochalasin B is a relatively specific and safe ocular penetration enhancer compared with other classical penetration enhancers such as bile salts, surfactants, etc.

Another strategy to improve paracellular transport is to make use of active transport systems. Active transport of glucose or amino acids, which is coupled to sodium transport, across the intestinal mucosa into the inter- cellular lateral spaces creates an osmotic force for fluid flow, and this in turns triggers contraction of the perijunctional actomyosin, resulting in increased paracellular permeability (decreased TEER) (20). Martinez- Palomo (21) showed that a hypertonic lysine solution induced a reversible opening of the tight junction of the toad urinary bladder without gross deformation of tight junctions. The decreased TEER was reversed when an isotonic solution was replaced on the apical side of the epithelium. Due to complete reversibility, it appears that increased paracellular trans- port by applying a hypertonic solution works in isolated tissues. However, a hypertonic solution may irritate the eye and induce tear production, which flushes away the applied drug. Therefore, the practicality of this approach is questionable and has yet to be confirmed.

296 Lee and Robinson

B. Enhanced Transcellular Transport Enhancers that increase transcellular permeability to drugs probably do so

by affecting membrane lipids and protein components. It is shown that fatty acids and their derivatives have been found to act primarily on the phos- pholipid component of membranes thereby creating disorder, resulting in increased permeability (22).

Membrane cholesterol is another target for enhanced transcellular delivery. It was postulated that extraction of cholesterol out of the epithelial membrane by medium chain monoglycerides, glyceryl-monooctanoate, gly- ceryl-1-monodecanoate, and glyceryl-monododecanoate promoted rectal cefoxitin absorption. However, some fatty acids act on the protein compo- nent in membranes. This is certainly the case for caprylate (22).

Nonprotein thios are another membrane component where certain enhancers can act. The good correlation between reduced nonprotein thiols and enhanced transport of hydrophilic compounds suggests an important role for nonprotein thiols in preventing the transport of hydrophilic com- pounds (22). Murakami et al. (23) showed that depletion of these nonpro- tein thiols by treating with SH-modifying agents like diethyl maleate, diethyl ethoxymethylenemalonate, ethanol, or alicylates enhanced the mucosal to serosal transport of many hydrophilic compounds including cefoxitin and phenol red in rat intestinal tissue.

V. NEW PENETRATION ENHANCERS Typically, classical penetration enhancers have a nonspecific action on bio-

logical membranes. They work by reversibly or permanently damaging the membranes so that their safety is questionable. Newer penetration enhan- cers have been introduced in ocular drug delivery recently with an aim to solving this problem.

A. Cyclodextrin Cyclodextrins are a group of homologous cyclic oligosaccharides consisting

of six, seven, and eight glucose units, namely, a-, b-, and g-cyclodextrin (Fig.

5) (24), respectively. Typically, cyclodextrins act as true carriers by keeping hydrophobic molecules in solution by their hydrophobic cores, i.e., com- plexation between hydrophobic molecules and the inner hydrophobic core of cyclodextrins. In other words, they are not capable of modifying the permeability of a biological barrier. On the other hand, drug absorption may be limited by the release of the drug from the drug-cyclodextrin com-

Ocular Penetration Enhancers 297

Figure 5 Structures of a-, b-, and g-cyclodextrins. (From Ref. 24.)

plex. As a result, addition of a cyclodextrin in an ophthalmic formulation does not necessarily increase the ocular bioavailability. It may adversely affect drug absorption (25,26).

However, a recent study showed that a-cyclodextrin has a significant penetration-enhancing effect (10-fold) on the corneal permeability of pilo- carpine (27). It was speculated that such an effect was achieved by direct interaction of the a-cyclodextrin with the corneal epithelium, which led to subsequent destabilization of the cell membrane since the same effect was also observed when the cornea was pretreated with the a-cyclodextrin before the transport study. Although a-cyclodextrin only produced minimal dama- ging effect on the cornea at low concentration (8%), the toxic effect at higher concentration (13% in this study) is still not known.

B. 1-Dodecylazacycloheptan-2-one (Azone 1 )

Although Azone 1 was extensively studied as a percutaneous penetration enhancer (28–30), its use in the ocular route is still in its infancy. The exact mechanism of the penetration enhancer effect is not known.

However, it can be speculated in terms of its lipophilicity (31). Azone 1 is highly lipophilic (log P>7), which can incorporate into lipoidal cell mem- brane and exert a penetration enhancing effect. It is interesting to note that 0.1% can enhance corneal penetration of hydrophilic compounds by at least 20-fold but inhibit corneal penetration of lipophilic compounds such as flurbiprofen. This was further supported by an earlier study that demon-

strated that Azone 1 did not improve the ocular bioavailability of levobu- nolol (32). It was speculated that insertion of Azone 1 into the corneal epithelium changed the structure and fluidity of the cell membrane.

298 Lee and Robinson Loosening of tight junctions and subsequent water (corneal swelling was

observed in the study) and drug influx might also be one of the possible mechanisms. It was believed that retardation of absorption of lipophilic molecules was due to creation of a more hydrated barrier, which retards entry of lipophilic molecules. However, this is a pure speculation, and further experiments need to be done to clarify the exact mechanism.

Safety is still the major concern in using Azone 1 as an ocular pene- tration enhancer. It is necessary to keep the Azone 1 concentration to a

minimum (<0.1%) since higher concentrations might cause ocular discom- fort, conjunctival hyperemia, and epithelial thinning as a result of erosion and/or atrophy (33). Fortunately, in vitro experiments showed that 0.1% was enough to enhance penetration for most of the compounds under inves- tigation to a significant extent (33).

C. Saponin Saponin is a type of polysaccharide isolated from the bark of the Quillaja

saponaria tree. Saponin is an amphiphilic compound that has surface activ- ity. As a result, although the exact mechanism of the penetration enhancing effect is not well understood, it is believed that the penetration enhancing effect relies solely on its detergent action. 0.5% Saponin increased corneal permeability of atenolol by two- to threefold but only slightly improved the delivery of relatively lipophilic timolol and befunolol. Saponin was also extensively studied as a penetration promoter for systemic delivery of macromolecules via the eye. Saponin demonstrated improvement in the systemic delivery of insulin and glucagon via the ocular route in various species (34–39). It was shown that the penetration-enhancing effect of sapo- nin was simply a direct detergent action since there did not exist a linear correlation between efficacy and surfactant strength. Therefore, the exact mechanism is still a mystery. The major concern in using saponin in an ophthalmic product is still safety. Concentrations higher than 0.5% are irritating to the eye (35).